US2024200062A1PendingUtilityA1
Methods of treating duchenne muscular dystrophy using peptide-oligonucleotide conjugates
Est. expiryMar 12, 2041(~14.6 yrs left)· nominal 20-yr term from priority
C12N 2310/3233C12N 2310/11C07K 7/08A61K 9/0019A61P 21/00A61K 47/64A61K 31/7105A61P 25/14C12N 15/113
47
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Claims
Abstract
Disclosed are methods of treating a subject having Duchenne muscular dystrophy. The method includes administration of 1 mg/kg to 60 mg/kg of a conjugate of an oligonucleotide and a peptide covalently bonded or linked via a linker to the oligonucleotide to the subject (e.g., a subject amenable to exon 51 skipping). The peptide including at least one cationic domain including at least 4 amino acid residues and at least one hydrophobic domain including at least 3 amino acid residues, provided that the peptide includes a total of 7 to 40 amino acid residues, and provided that the at least one cationic domain includes a beta-alanine residue in combination with arginine and/or histidine residues. The oligonucleotide including a total of 12 to 40 contiguous nucleobases, wherein at least 12 contiguous nucleobases are complementary to a target sequence in a human dystrophin gene.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method of treating a subject having Duchenne muscular dystrophy, the method comprising administering 1 mg/kg to 60 mg/kg of a conjugate of an oligonucleotide and a peptide covalently bonded or linked via a linker to the oligonucleotide,
the peptide comprising at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, provided that the peptide comprises a total of 7 to 40 amino acid residues, and provided that the at least one cationic domain comprises a beta-alanine residue in combination with arginine and/or histidine residues; and the oligonucleotide comprising a total of 12 to 40 contiguous nucleobases, wherein at least 12 contiguous nucleobases are complementary to a target sequence in a human dystrophin gene.
2 . The method of claim 1 , wherein the oligonucleotide comprises a sequence selected from the group consisting of:
(SEQ ID NO: 106)
5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′;
or
(SEQ ID NO: 107)
5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′;
(SEQ ID NO: 108;
5′-CUCAUACCUUCUGCUUGAUGAUC-3′;
(SEQ ID NO: 109)
5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′;
(SEQ ID NO: 110)
5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′;
(SEQ ID NO: 111)
5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′;
(SEQ ID NO: 112)
5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′;
(SEQ ID NO: 113)
5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′;
(SEQ ID NO: 114)
5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′;
(SEQ ID NO: 115)
5′-CACCCACCAUCACCCUCUGUG-3′;
(SEQ ID NO: 116)
5′-AUCAUCUCGUUGAUAUCCUCAA-3′;
and their thymine-substitution analogues
3 . The method of claim 1 , wherein each cationic domain has length of between 4 and 12 amino acid residues.
4 . The method of claim 3 , wherein each cationic domain has length of between 4 and 7 amino acid residues.
5 . The method of any one of claims 1-4 , wherein each cationic domain comprises at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids.
6 . The method of any one of claims 1-4 , wherein each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, at least 70% arginine and/or histidine residues.
7 . The method of any one of claims 1-4 , wherein each cationic domain comprises one of the following sequences: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11 ), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19) or any combination thereof.
8 . The method of any one of claims 1-4 , wherein each cationic domain comprises or consists of one the following sequences: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO:
11 . , RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19) or any combination thereof.
9 . The method of any one of claims 1-4 , wherein the peptide comprises two cationic domains.
10 . The method of any one of claims 1-4 , wherein each hydrophobic domain has a length of 3 to 6 amino acids, preferably each hydrophobic domain has a length of 5 amino acids.
11 . The method of any one of claims 1-4 , wherein each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.
12 . The method of any one of claims 1-4 , wherein each hydrophobic domain comprises phenylalanine, leucine, Isoleucine, tyrosine, tryptophan, proline, and/or glutamine residues; preferably wherein each hydrophobic domain comprises or consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and/or glutamine residues.
13 . The method of any one of claims 1-4 , wherein the peptide comprises one hydrophobic domain.
14 . The method of any one of claims 1-4 , wherein the or each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26) or any combination thereof.
15 . The method of any one of claims 1-4 , wherein the or each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26) or any combination thereof.
16 . The method of any one of claims 1-4 , wherein the peptide comprises or consists of two cationic domains and one hydrophobic domain.
17 . The method of any one of claims 1-4 , wherein the peptide comprises or consists of one hydrophobic core domain flanked by two cationic arm domains.
18 . The method of any one of claims 1-4 , wherein the peptide comprises or consists of one hydrophobic core domain comprising a sequence selected from: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26), flanked by two cationic arm domains each comprising a sequence selected from: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO:
11 . , RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19).
19 . The method of any one of claims 1-4 , wherein the peptide comprises or consists of one of the following sequences: RBRRBRRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRYQFLIRBRBR (SEQ ID NO:
31 . , RBRRBRRILFQYRBRBR (SEQ ID NO: 32), RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), RBRRBRRFQILYHBHBR (SEQ ID NO: 38), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).
20 . The method of any one of claims 1-4 , wherein the peptide has the following amino acid sequence RBRRBRFQILYBRBR (SEQ ID NO: 35).
21 . The method of any one of claims 1-4 , wherein the peptide has the following amino acid sequence RBRRBRRFQILYRBHBH (SEQ ID NO: 37).
22 . The method of any one of claims 1-4 , wherein the peptide has the following amino acid sequence RBRRBRFQILYRBHBH (SEQ ID NO: 44).
23 . The method of any one of claims 1-4 , wherein the peptide is bonded to the rest of the conjugate through its N-terminus.
24 . The method of claim 23 , wherein the C-terminus of the peptide is —NH 2 .
25 . The method of any one of claims 1-4 , wherein the peptide is bonded to the rest of the conjugate through its C-terminus.
26 . The method of claim 25 , wherein the peptide is acylated at its N-terminus.
27 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
[peptide]-[linker]-[oligonucleotide]
28 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
29 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
[peptide]-[linker]-[peptide]-[linker]-[oligonucleotide].
30 . The method of any one of claims 1-4 , wherein each linker is independently of formula (I):
T 1 —(CR 1 R 2 ) n —T 2 . (I)
wherein
T 1 is a divalent group for attachment to the peptide and is selected from the group consisting of —NH— and carbonyl;
T 2 is a divalent group for attachment to an oligonucleotide and is selected from the group consisting of —NH- and carbonyl;
n is 1, 2 or 3;
each R 1 is independently —Y 1 —X 1 —Z 1 ,
wherein
Y 1 is absent or —(CR A1 R A2 ) m —, wherein m is 1, 2, 3 or 4, and R A1 and R A2 are each independently hydrogen, OH, or (1-2C)alkyl;
X 1 is absent, —O—, —C(O)—, —C(O)O—, —OC(O)—, —CH(OR A3 )—, —N(R A3 )—, —N(R A3 )—C(O)—, —N(R A3 )—C(O)O—, —C(O)—N(R A3 )—, —N(R A3 )C(O)N(R A3 )—, —N(R A3 )C(N R A3 )N(R A3 )—, —SO—, —S—, —SO 2 —, —S(O) 2 N(R A3 )—, or —N(R A3 )SO 2 —, wherein each R A3 is independently selected from hydrogen and methyl; and
Z 1 is a further oligonucleotide or is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl,
wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 , and (1-4C)alkoxy, wherein R A4 and R A5 are each independently selected from the group consisting of hydrogen and (1-4C)alkyl; and
each R 2 is independently —Y 2 —X 2 —Z 2 , wherein
Y 2 is absent or a group of the formula —[CR B1 R B2 ] n — in which m is an integer selected from 1, 2, 3 or 4, and R B1 and R B2 are each independently selected from hydrogen, OH or (1-2C)alkyl;
X 2 is absent, —O—, —C(O)—, —C(O)O—, —OC(O)—, —CH(OR B3 )—, —N(R B3 )—, —N(R B3 )—C(O)—, —N(R B3 )—C(O)O—, —C(O)—N(R B3 )—, —N(R B3 )C(O)N(R B3 )—, —N(R B3 )C(NR B3 )N(R B3 )—, —SO—, —S— —SO 2 —, —S(O) 2 N(R B3 )—, or —N(R B3 )—SO 2 —, wherein each R B3 is independently selected from hydrogen or methyl; and
Z 2 is selected from hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, wherein each (1-6C)alkyl, (2- 6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR B4 R B5 , and (1-4C)alkoxy, wherein R B4 and R B5 are each independently hydrogen or (1-2C)alkyl; with the proviso that; when n=1 and T 1 and T 2 are different to one another, then R 1 and R 2 are not both H; when n=1, T 1 and T 2 are different to one another and one of R 1 and R 2 is H then the other of R 1 and R 2 is not methyl; or when n=2 and each occurrence of R 1 and R 2 is H, then T 1 and T 2 are both —C(O)- or are both —NH—.
31 . The method of claim 30 , wherein T 2 is —C(O)—.
32 . The method of claim 30 , wherein each R 1 is independently —Y 1 —X 1 —Z 1 , wherein:
Y 1 is absent or —(CR A1 R A2 ) m —, wherein m is 1, 2, 3 or 4, and R A1 and R A2 are each hydrogen or (1-2C)alkyl;
X 1 is absent, —O—, —C(O)- 7 —C(O)O—, —N(R A3 )—, —N(R A3 )—C(O)—, —C(O)—N(R A3 )—, —N(R A3 )C(O)N(R A3 )—, —N(R A3 )C(N R A3 )N(R A3 )- or—S—, wherein each R A3 is independently hydrogen or methyl; and
Z 1 is a further oligonucleotide or is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NR A4 R A5 , and (1-4C)alkoxy, wherein RM and R A5 are each independently hydrogen or (1-2C)alkyl.
33 . The method of claim 30 , wherein each R 1 is independently —Y 1 —X 1 —Z 1 , wherein:
Y 1 is absent or —(CR A1 R A2 ) m —, wherein m is 1, 2, 3, or 4, and R A1 and R″ are each independently hydrogen or (1-2C)alkyl;
X 1 is absent, —O—, —C(O)—, —C(O)O—, —N(R A3 )—, —N(R A3 )—C(O)—, —C(O)—N(R A3 )—, —N(R A3 )C(O)N(R A3 )—, —N(R A3 )C(NR A3 )N(R A3 )—, or—S—, wherein each R A3 is independently hydrogen or methyl; and
Z 1 is a further oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.
34 . The method of claim 30 , wherein each R 1 is independently 7 wherein:
Y 1 is absent or a group of the formula —(cRA1RA2) m —, wherein m is 1, 2, 3 or 4, and R A1 and R A2 are each independently hydrogen or (1-2C)alkyl; X 1 is absent, —C(O)—, —C(O)O—, —N(R A3 )—C(O)—, —C(O)—N(R A3 )—, wherein each R A3 is hydrogen or methyl; and Z 1 is a further oligonucleotide or is hydrogen, (1- 6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (1-6C)alkyl, aryl, (3- 6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.
35 . The method of claim 30 , wherein each R 1 is independently —Y 1 —X 1 —Z 1 , wherein:
Y 1 is absent, —(CH 2 )—, or —(CH 2 CH 2 )—;
X 1 is absent, —N(R A3 )—C(O)—, —C(O)—N(R A3 )—, wherein each R A3 is independently hydrogen or methyl; and
Z 1 is hydrogen or (1-2C)alkyl.
36 . The method of claim 30 , wherein each R 2 is independently —Y 2 —Z 2 ,
wherein Y 2 is absent or —(CR B1 R B2 R B2 ) m—, wherein m is 1, 2, 3 or 4, and R B1 and R B2 are each independently hydrogen or (1-2C)alkyl; and
Z 2 is hydrogen or (1-6C)alkyl.
37 . The method of claim 30 , wherein each R 2 is hydrogen.
38 . The method of claim 30 , wherein n is 2 or 3.
39 . The method of claim 30 , wherein n is 1.
40 . The method of any one of claims 1-4 , wherein the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and gamma-aminobutyric acid residues.
41 . The method of any one of claims 1-4 , wherein the linker is of the following structure:
42 . The method of any one of claims 1-4 , wherein the linker is of the following structure:
43 . The method of any one of claims 1-4 , wherein the linker is of the following structure:
44 . The method of any one of claims 1-4 , wherein the linker is of the following structure:
45 . The method of any one of claims 1-4 , wherein the linker is of the following structure:
46 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
47 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
48 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
49 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
50 . The method of any one of claims 1-4 , wherein the conjugate comprises or is of the following structure:
51 . The method of any one of claims 1-4 , wherein the oligonucleotide is bonded to the linker or the peptide at its 3′ terminus.
52 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
53 . The method of claim 52 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
54 . The method of claim 52 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
55 . The method of claim 52 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
56 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to N-terminus of peptide RBRRBRFQILYBRBR—NH 2 , wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
57 . The method of claim 56 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via a glutamic acid residue to N-terminus of peptide RBRRBRFQILYBRBR—NH 2 , wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
58 . The method of claim 56 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to N-terminus of peptide RBRRBRFQILYBRBR—NH 2 , wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
59 . The method of claim 56 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to N-terminus of peptide RBRRBRFQILYBRBR—NH 2 , wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
60 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via gamma-aminobutyric acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
61 . The method of claim 60 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via gamma-aminobutyric acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
62 . The method of claim 60 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via gamma-aminobutyric acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
63 . The method of claim 60 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via gamma-aminobutyric acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
64 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
65 . The method of claim 64 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
66 . The method of claim 64 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH, wherein free —COOH, if any, in the glutamic acid residue is replaced with —CONH 2 .
67 . The method of claim 64 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a glutamic acid residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH, wherein free —COON, if any, in the glutamic acid residue is replaced with —CONH 2 .
68 . The method of any one of claim 1-4 or 52-67 , wherein the linker is of the following structure:
69 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR.
70 . The method of claim 69 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR.
71 . The method of claim 69 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR.
72 . The method of claim 69 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYBRBR.
73 . The method of claim 1 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106), 5′-ACCAGAGUAACAGUCUGAGUAGGAGC-3′ (SEQ ID NO: 107), 5′-CUCAUACCUUCUGCUUGAUGAUC-3′ (SEQ ID NO: 108), 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), 5′-ACAUCAAGGAAGAUGGCAUUUCUAGUUUGG-3′ (SEQ ID NO: 110), 5′-ACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 111), 5′-CUCCAACAUCAAGGAAGAUGGCAUUUCUAG-3′ (SEQ ID NO: 112), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAG-3′ (SEQ ID NO: 113), 5′-AUCAUUUUUUCUCAUACCUUCUGCUAGGAGCUAAAAAG-3′ (SEQ ID NO: 114), 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or 5′-AUCAUCUCGUUGAUAUCCUCAA-3′ (SEQ ID NO: 116), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
74 . The method of claim 73 , wherein the conjugate comprises or consists of 5′-CTCCAACATCAAGGAAGATGGCATTTCTAG-3′ (SEQ ID NO: 106) having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
75 . The method of claim 73 , wherein the conjugate comprises or consists of 5′-UUCUGUCCAAGCCCGGUUGAAAUC-3′ (SEQ ID NO: 109), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
76 . The method of claim 73 , wherein the conjugate comprises or consists of 5′-CACCCACCAUCACCCUCUGUG-3′ (SEQ ID NO: 115), or a thymine-substitution analogue thereof, having a 3′-terminus covalently linked via a beta-alanine residue to C-terminus of peptide Ac-RBRRBRFQILYRBHBH.
77 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the oligonucleotide is a morpholino.
78 . The method of claim 77 , wherein all morpholino internucleoside linkages are —P(O)(NMe 2 )O—.
79 . The method of claim 78 , wherein the oligonucleotide comprises the following group as its 5′ terminus:
80 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the oligonucleotide comprises the following group as its 5′ terminus:
81 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the conjugate is administered parenterally.
82 . The method of claim 81 , wherein the conjugate is administered by infusion.
83 . The method of claim 82 , wherein the conjugate is administered by intravenous infusion.
84 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the subject is amenable to exon 51 skipping.
85 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the conjugate is administered to the subject at a frequency that is weekly to quarterly.
86 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the conjugate is administered to the subject at a frequency that is weekly to monthly.
87 . The method of claim 86 , wherein the frequency is weekly, biweekly, or monthly.
88 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the frequency is quarterly.
89 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein 40 mg/kg to 60 mg/kg, 30 mg/kg to 50 mg/kg, 30 mg/kg to 40 mg/kg, 40 mg/kg to 50 mg/kg, 50 mg/kg to 60 mg/kg, 35 mg/kg to 45 mg/kg, 45 mg/kg to 55 mg/kg, 35 mg/kg to 55 mg/kg, 30 mg/kg to 45 mg/kg, 35 mg/kg to 50 mg/kg, 40 mg/kg to 55 mg/kg, 45 mg/kg to 60 mg/kg, 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 5 mg/kg to 25 mg/kg, 10 mg/kg to 30 mg/kg, 1 mg/kg to 15 mg/kg, 5 mg/kg to 20 mg/kg, 10 mg/kg to 25 mg/kg, 15 mg/kg to 30 mg/kg, 1 mg/kg to 10 mg/kg, 5 mg/kg to 15 mg/kg, 10 mg/kg to 20 mg/kg, 15 mg/kg to 25 mg/kg, 20 mg/kg to 30 mg/kg, 1 mg/kg to 25 mg/kg, 4 mg/kg to 20 mg/kg, 6 mg/kg to 15 mg/kg, or 8 mg/kg to 10 mg/kg of conjugate is administered.
90 . The method of claim any one of claims 1-4, 52-67, and 69-76 , wherein 1 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, or 60 mg/kg of conjugate is administered.
91 . The method of claim 90 , wherein 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg of conjugate is administered.
92 . The method of any one of claims 1-4, 52-67, and 69-76 , wherein the method is for use in treating a cardiac effect of DMD including, e.g., cardiomyopathy (e.g., dilated, hypertrophic, or restrictive cardiomyopathy), heart failure, and/or a cardiac arrhythmia.Join the waitlist — get patent alerts
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